Rapid identification of cytochrome P450cam variants by in vivo screening of active site libraries

Speight, Robert E., Hancock, Fred E., Winkel, Chris, Bevinakatti, Hanamanthsa S., Sarkar, Manish, Flitsch, Sabine L., & Turner, Nicholas J. (2004) Rapid identification of cytochrome P450cam variants by in vivo screening of active site libraries. Tetrahedron: Asymmetry, 15(18), pp. 2829-2831.

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The selection of cytochrome P450 enzymes from large variant libraries, and the subsequent use of these enzymes in preparative scale biotransformations, remains a formidable challenge due to the complexities of the associated electron transport systems. Here, a powerful approach for the generation and screening of P450cam libraries for new function is presented that is both flexible and robust. A targeted library was generated wherein only the P450cam active-site amino acids Y96 and F98 were fully randomized and biotransformations, using a novel P450cam whole-cell system, were screened by GC–MS for the hydroxylation of diphenylmethane. One in 50 of the reactions screened, including 16 different variants, produced 4-hydroxydiphenylmethane with up to 92% conversion observed in the case of the Y96A variant. These results demonstrate a primary example of the screening of P450cam libraries in a format that is compatible with extension to preparative scale reactions.

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ID Code: 70986
Item Type: Journal Article
Refereed: Yes
Additional URLs:
DOI: 10.1016/j.tetasy.2004.06.053
ISSN: 1362-511X
Divisions: Current > Schools > School of Chemistry, Physics & Mechanical Engineering
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Deposited On: 04 May 2014 22:55
Last Modified: 04 May 2014 22:55

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